EPR analysis of cyanide complexes of wild-type human neuroglobin and mutants in comparison to horse heart myoglobin

- First reported EPR study of cyanide-ligated neuroglobins.
- First determination of the full 13C hyperfine tensor of the cyanide carbon in a cyanide-ligated globin.
- Disagreement of 13C hyperfine data of cyanide-ligated myoglobin with predictions from NMR reveal flaws in theortical model.
- The present 13C hyperfine data challenge the current interpretation of 13C NMR shifts in cyanide-ligated haem proteins.

Electron paramagnetic resonance (EPR) data reveal large differences between the ferric (13C-)cyanide complexes of wild-type human neuroglobin (NGB) and its H64Q and F28L point mutants and the cyanide complexes of mammalian myo- and haemoglobin. The point mutations, which involve residues comprising the distal haem pocket in NGB, induce smaller, but still significant changes, related to changes in the stabilization of the cyanide ligand. Furthermore, for the first time, the full 13C hyperfine tensor of the cyanide carbon of cyanide-ligated horse heart myoglobin (hhMb) was determined using Davies ENDOR (electron nuclear double resonance). Disagreement of these experimental data with earlier predictions based on 13C NMR data and a theoretical model reveal significant flaws in the model assumptions. The same ENDOR procedure allowed also partial determination of the corresponding 13C hyperfine tensor of cyanide-ligated NGB and H64QNGB. These 13C parameters differ significantly from those of cyanide-ligated hhMb and challenge our current theoretical understanding of how the haem environment influences the magnetic parameters obtained by EPR and NMR in cyanide-ligated haem proteins.